Nucleic acids encoding polypeptides having proteolytic activity

ABSTRACT

The present invention relates to isolated nucleic acid sequences encoding polypeptides having proteolytic activity. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the nucleic acid sequences as well as recombinant methods for producing the polypeptides.

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims, under 35 U.S.C. 119, priority or thebenefit of Danish application no. PA 2000 01455 filed Oct. 2, 2000 andU.S. provisional application No. 60/239,064 filed Oct. 6, 2000, thecontents of which are fully incorporated herein by reference.

FIELD OF THE INVENTION

[0002] The present invention relates to isolated nucleic acid sequencesencoding polypeptides having proteolytic activity. The invention alsorelates to nucleic acid constructs, vectors, and host cells comprisingthe nucleic acid sequences as well as recombinant methods for producingthe polypeptides.

BACKGROUND OF THE INVENTION

[0003] In the detergent industry enzymes have for more than 30 yearsbeen implemented in washing formulations. Enzymes used in suchformulations comprise proteases, lipases, amylases, cellulases, as wellas other enzymes, or mixtures thereof. Commercially most importantenzymes are proteases.

[0004] Detergent proteases have been developed by isolation of proteasesfound in nature followed by testing in detergent formulations. Mostdetergent proteases are obtained from members of the genus Bacillus.

[0005] Examples of commercial protease products are Alcalase®, Esperase®and Savinase®, all supplied by Novo Nordisk A/S, Denmark. These andsimilar enzyme products from other commercial sources are active indetergent solutions, i.e. at pH values in the range of from 8 to 11 andin the presence of sequestering agents, surfactants and bleaching agentssuch as sodium borate. The Alcalase® protease is produced by strains ofthe species Bacillus licheniformis. The Esperase® and Savinase®proteases are obtained by cultivation of strains of alkalophilicBacilli.

[0006] WO 92117577 describes a protease isolated from Bacillus sp.TY145, NCIMB 40339. The isolated protease had a pH optimum in the rangeof from 8 to 11, a temperature optimum in the range of from 45 to 55 C.,a pi around 8.8, and an apparent molecular weight of about 38 kD. Thegene producing the above-mentioned protease has now been cloned andexpressed in Bacillus subtilis. Thus, it is an object of the presentinvention to provide isolated nucleic acid sequences encodingpolypeptides having proteolytic activity a well as variants of saidprotease.

SUMMARY OF THE INVENTION

[0007] In a first aspect, the present invention relates to an isolatednucleic acid sequence encoding a polypeptide having proteolyticactivity, selected from the group consisting of:

[0008] (a) a nucleic acid sequence encoding a polypeptide having anamino acid sequence which has at least 75% identity with amino acids 1to 311 of SEQ ID NO: 2;

[0009] (b) a nucleic acid sequence having at least 70% identity withnucleotides 371 to 1303 of SEQ ID NO: 1;

[0010] (c) a nucleic acid sequence, which hybridizes under lowstringency conditions with

[0011] (i) the nucleic acid sequence of SEQ ID NO: 1,

[0012] (ii) a subsequence of (i) of at least 100 nucleotides, or

[0013] (iii) a complementary strand of (i) or (ii);

[0014] (d) an allelic variant of (a), (b), or (c); and

[0015] (e) a subsequence of (a), (b), (c), or (d), wherein thesubsequence encodes a polypeptide fragment which has proteolyticactivity.

[0016] In a second aspect, the present invention relates to a variant ofthe polypeptide having the amino acid sequence shown as amino acids 1 to311 of SEQ ID NO: 1, which comprises at least one modification comparedto amino acids 1 to 311 of SEQ ID NO: 1 and which has at least 75%identity with amino acids 1 to 311 of SEQ ID NO: 1.

[0017] In other aspects, the present invention also relates to detergentcompositions comprising such variants, nucleic acid constructs, vectors,and host cells comprising the nucleic acid sequences as well asrecombinant methods for producing the polypeptides.

BRIEF DESCRIPTION OF THE FIGURES

[0018]FIG. 1 shows the relation between temperature and proteolyticactivity of the polypeptide having the amino acid sequence shown asamino acids 1 to 311 of SEQ ID NO: 1 (the polypeptide being obtainedaccording to Example 1, with casein as a substrate and at pH 9.5).

[0019]FIG. 2 shows the relation between pH and proteolytic activity ofthe polypeptide having the amino acid sequence shown as amino acids 1 to311 of SEQ ID NO: 1 (the polypeptide being obtained according to Example1, with casein as substrate and at 25° C.).

DETAILED DESCRIPTION OF THE INVENTION

[0020] Isolated Nucleic Acid Sequences Encoding Polypeptides HavingProteolytic Activity

[0021] The term “proteolytic activity” is defined herein as thecapability of the polypeptide to catalyze the hydrolysis of peptidebonds. For purposes of the present invention, proteolytic activity isexpressed in Casein Protease Units (CPU), where one unit CPU is definedas the amount of polypeptide liberating 1 mM of primary amine groups(determined by comparison to a serine standard) per minute understandard conditions, i.e. incubation for 30 minutes at 25° C. and pH9.5. A folder AF 222, describing the analytical method in furtherdetails, is available upon request to Novo Nordisk A/S, which folder ishereby included by reference.

[0022] The term “isolated nucleic acid sequence” as used herein refersto a nucleic acid sequence which is essentially free of other nucleicacid sequences, e.g., at least about 20% pure, preferably at least about40% pure, more preferably at least about 60% pure, even more preferablyat least about 80% pure, and most preferably at least about 90% pure asdetermined by agarose electrophoresis. For example, an isolated nucleicacid sequence can be obtained by standard cloning procedures used ingenetic engineering to relocate the nucleic acid sequence from itsnatural location to a different site where it will be reproduced. Thecloning procedures may involve excision and isolation of a desirednucleic acid fragment comprising the nucleic acid sequence encoding thepolypeptide, insertion of the fragment into a vector molecule, andincorporation of the recombinant vector into a host cell where multiplecopies or clones of the nucleic acid sequence will be replicated. Thenucleic acid sequence may be of genomic, cDNA, RNA, semi synthetic,synthetic origin, or any combinations thereof.

[0023] In a first embodiment, the present invention relates to isolatednucleic acid sequences encoding polypeptides having an amino acidsequence which has a degree of identity to amino acids 1 to 311 of SEQID NO: 2 (i.e., the mature polypeptide) of at least 75%, preferably atleast 80%, more preferably at least 90%, even more preferably at least95%, most preferably at least 97%, and even most preferably at least99%, which have proteolytic activity (hereinafter “homologouspolypeptides”). In a preferred embodiment, the homologous polypeptideshave an amino acid sequence which differs by five amino acids,preferably by four amino acids, more preferably by three amino acids,even more preferably by two amino acids, and most preferably by oneamino acid from amino acids 1 to 311 of SEQ ID NO: 2. For purposes ofthe present invention, the degree of identity between two amino acidsequences is determined, e.g., by the Clustal method (Higgins, 1989,CABIOS 5: 151-153) using the LASERGENE™ MEGALIGN™ software (DNASTAR,Inc., Madison, Wis.) with an identity table and the following multiplealignment parameters: Gap penalty of 10, and gap length penalty of 10.Pairwise alignment parameters were Ktuple=1, gap penalty=3, windows=5,and diagonals=5.

[0024] Preferably, the nucleic acid sequence of the present inventionencodes a polypeptide that comprises amino acids 1 to 311 of SEQ ID NO:2, which is the mature polypeptide of SEQ 5 ID NO: 2, or an allelicvariant thereof; or a fragment thereof that has proteolytic activity. Inanother preferred embodiment, the nucleic acid sequence of the presentinvention encodes a polypeptide that consists of amino acids 1 to 311 ofSEQ ID NO: 2 or an alletic variant thereof; or a fragment thereof thathas proteolytic activity.

[0025] The present invention also encompasses nucleic acid sequencesthat encode a 10 polypeptide having the amino acid sequence of SEQ IDNO: 2, which differ from SEQ ID NO: 1 by virtue of the degeneracy of thegenetic code. The present invention also relates to subsequences of SEQID NO: 1 that encode fragments of SEQ ID NO: 2 that have proteolyticactivity.

[0026] A subsequence of SEQ ID NO: 1 is a nucleic acid sequenceencompassed by SEQ ID NO: 1 except that one or more nucleotides from the5′ and/or 3′ end have been deleted.

[0027] A fragment of SEQ ID NO: 2 is a polypeptide having one or moreamino acids deleted from the amino and/or carboxy terminus of this aminoacid sequence.

[0028] An allelic variant denotes any of two or more alternative formsof a gene occupying the same chromosomal locus. Allelic variation arisesnaturally through mutation, and may result in polymorphism withinpopulations. Gene mutations can be silent (no change in the encodedpolypeptide) or may encode polypeptides having altered amino acidsequences. The allelic variant of a polypeptide is a polypeptide encodedby an allelic variant of a gene.

[0029] The amino acid sequences of the homologous polypeptides maydiffer from the amino acid sequence of SEQ ID NO: 2 or the maturepolypeptide thereof by an insertion or deletion of one or more aminoacid residues and/or the substitution of one or more amino acid residuesby different amino acid residues. Preferably, amino acid changes are ofa minor nature, that is conservative amino acid substitutions that donot significantly affect the folding and/or activity of the protein;small deletions, typically of one to about 30 amino acids; small amino-or carboxyl-terminal extensions, such as an amino-terminal methionineresidue; a small linker peptide of up to about 20-25 residues; or asmall extension that facilitates purification by changing net charge oranother function, such as a poly-histidine tract, an antigenic epitopeor a binding domain.

[0030] Examples of conservative substitutions are within the group ofbasic amino acids (such as arginine, lysine and histidine), acidic aminoacids (such as glutamic acid and aspartic acid), polar amino acids (suchas glutamine and asparagine), hydrophobic amino acids (such as leucine,isoleucine, methionine and valine), aromatic amino acids (such asphenylalanine, tryptophan and tyrosine), and small amino acids (such asglycine, alanine, serine and threonine). Amino acid substitutions, whichdo not generally alter the specific activity, are known in the art andare described, for example, by H. Neurath and R. L. Hill, 1979, In, TheProteins, Academic Press, New York. The most commonly occurringexchanges are Ala/Ser, Val/Ile, Asp/Glu, Thr/Ser, Ala/Gly, Ala/Thr,Ser/Asn, AlaNal, Ser/Gly, Tyr/Phe, Ala/Pro, Lys/Arg, Asp/Asn, Leu/Ile,LeuNal, Ala/Glu, and Asp/Gly as well as these in reverse.

[0031] In a second embodiment, the present invention relates to isolatednucleic acid sequences which have a degree of identity to the maturepolypeptide coding sequence of SEQ 10 ID NO: 1 (i.e., nucleotides 371 to1303) of at least 70%, preferably at least 80%, such as at least 90%,more preferably at least 95%, even more preferably at least 97%, andmost preferably at least 99% identity, which encode an activepolypeptide; or allelic variants and subsequences of SEQ ID NO: 1 whichencode polypeptide fragments which have proteolytic activity.Preferably, the nucleic acid sequence of the present invention comprisesthe nucleotides 371 to 1303 of SEQ ID NO: 1. In another preferredembodiment, the nucleic acid sequence of the present invention consistsof the nucleotides 371 to 1303 of SEQ ID NO: 1.

[0032] For purposes of the present invention, the degree of identitybetween two nucleic acid sequences is determined by, e.g., theWilbur-Lipman method (Wilbur and Lipman, 1983, Proceedings of theNational Academy of Science USA 80: 726-730) using the LASERGENE™MEGALIGN™ software (DNASTAR, Inc., Madison, Wis.) with an identity tableand the following multiple alignment parameters: Gap penalty of 10, andgap length penalty of 10. Pairwise alignment parameters were Ktuple=3,gap penalty=3, and windows=20.

[0033] By performing such alignments as described above, the followingidentities between SEQ ID NO: 1, SEQ ID NO: 2 and various knownproteases were found:

[0034] Percent identity between the polypeptide having the amino acidsequence shown as amino acids 1 to 311 of SEQ ID NO: 2 and APRSPHR¹⁾:72.8%, TA39²⁾: 65.2%, Subtilisin BPN′: 33.8%, Savinase ®: 34.6%.

[0035] Percent identity between the nucleic acid sequence shown asnucleotides 371 to 1303 of SEQ ID NO: 1 and the gene encoding APRSPHR¹⁾:68.3%, TA39²⁾: 61.0%, Subtilisin BPN′: 42.4%, Savinase ®: 40.7%.

[0036] In a third embodiment, the present invention relates to isolatednucleic acid sequences encoding polypeptides having proteolytic activitywhich hybridize under very low stringency conditions, preferably lowstringency conditions, more preferably medium stringency conditions,more preferably medium-high stringency conditions, even more preferablyhigh stringency conditions, and most preferably very high stringencyconditions with a nucleic acid probe which hybridizes under the sameconditions with (i) the nucleic acid sequence of SEQ ID NO: 1, (ii) asubsequence of (i), or (iii) a complementary strand of (i) or (ii) (see:J. Sambrook, E. F. Fritsch, and T. Maniatis, 1989, Molecular Cloning, ALaboratory Manual, 2d edition, Cold Spring Harbor, N.Y.). Thesubsequence of SEQ ID NO: 1 may be at least 100 nucleotides orpreferably at least 200 nucleotides. Moreover, the subsequence mayencode a polypeptide fragment, which has proteolytic activity.

[0037] The nucleic acid sequence of SEQ ID NO: 1 or a subsequencethereof, as well as the amino acid sequence of SEQ ID NO: 2 or afragment thereof, may be used to design a nucleic acid probe to identifyand clone DNA encoding polypeptides having proteolytic activity fromstrains of different genera or species according to methods well knownin the art. In particular, such probes can be used for hybridizationwith the genomic or cDNA of the genus or species of interest, followingstandard Southern blotting procedures, in order to identify and isolatethe corresponding gene therein. Such probes can be considerably shorterthan the entire sequence, but should be at least 15, preferably at least25, and more preferably at least 35 nucleotides in length. Longer probescan also be used. Both DNA and RNA probes can be used. The probes aretypically labeled for detecting the corresponding gene (for example,with ³²P, ³H, ³⁵S, biotin, or avidin). Such probes are encompassed bythe present invention.

[0038] Thus, a genomic DNA or cDNA library prepared from such otherorganisms may be screened for DNA that hybridizes with the probesdescribed above and which encodes a polypeptide having proteolyticactivity. Genomic or other DNA from such other organisms may beseparated by agarose or polyacrylamide gel electrophoresis, or otherseparation techniques. DNA from the libraries or the separated DNA maybe transferred to and immobilized on nitrocellulose or other suitablecarrier material. In order to identify a clone or DNA that is homologouswith SEQ ID NO: 1 or a subsequence thereof, the carrier material is usedin a Southern blot. For purposes of the present invention, hybridizationindicates that the nucleic acid sequence hybridizes to a nucleic acidprobe corresponding to the nucleic acid sequence shown in SEQ ID NO: 1,its complementary strand, or a subsequence thereof, under very low tovery high stringency conditions. Molecules to which the nucleic acidprobe hybridizes under these conditions are detected using X-ray film.

[0039] For long probes of at least 100 nucleotides in length, very lowto very high stringency conditions are defined as prehybridization andhybridization at 42° C. in 5× SSPE, 0.3% SDS, 200 μg/ml sheared anddenatured salmon sperm DNA, and either 25% formamide for very low andlow stringencies, 35% formamide for medium and medium-high stringencies,or 50% formamide for high and very high stringencies, following standardSouthern blotting procedures.

[0040] For long probes of at least 100 nucleotides in length, thecarrier material is finally washed three times each for 15 minutes using2× SSC, 0.2% SDS preferably at least at 45° C. (very low stringency),more preferably at least at 50° C. (low stringency), more preferably atleast at 55° C. (medium stringency), more preferably at least at 60° C.(medium-high stringency), even more preferably at least at 65° C. (highstringency), and most preferably at least at 70° C. (very highstringency).

[0041] For short probes which are about 15 nucleotides to about 70nucleotides in length, stringency conditions are defined asprehybridization, hybridization, and washing post-hybridization at 5° C.to 10° C. below the calculated T_(m) using the calculation according toBolton and McCarthy (1962, Proceedings of the National Academy ofSciences USA 48:1390) in 0.9 M NaCl, 0.09 M Tris-HCl pH 7.6, 6 mM EDTA,0.5% NP-40, 1× Denhardt's solution, 1 mM sodium pyrophosphate, 1 mMsodium monobasic phosphate, 0.1 mM ATP, and 0.2 mg of yeast RNA per mlfollowing standard Southern blotting procedures.

[0042] For short probes that are about 15 nucleotides to about 70nucleotides in length, the carrier material is washed once in 6× SCCplus 0.1% SDS for 15 minutes and twice each for 15 minutes using 6× SSCat 5° C. to 10° C. below the calculated T_(m).

[0043] In a fourth embodiment, the present invention relates to isolatednucleic acid sequences encoding polypeptides with proteolytic activityhaving the following physicochemical properties:

[0044] (a) pH optimum in the range of from pH 8 to 11 (at 25° C.),

[0045] (b) temperature optimum in the range of from 45 C. to 55 C. (atpH 9.5),

[0046] (c) immunochemical properties identical or partially identical tothose of a protease derived from Bacillus sp. TY145, NCIMB No. 40339.

[0047] The encoded polypeptide has a temperature optimum in the range offrom 45° C. to 55° C., preferably about 50° C., and a pH optimum in therange of from 8 to 11, preferably about 10.

[0048] The immunochemical properties can be determined immunologicallyby cross-reaction identity tests. The identity tests can be performed bythe well-known Ouchterlony double immunodiffusion procedure or by tandemcrossed immunoelectrophoresis according to N. H. Axelsen: Handbook ofImmunoprecipitation-in-gel Techniques, Blackwell Scientific Publications(1983), chapters 5 and 14. The terms “antigenic identity” and “partialantigenic identity” are described in the same book, chapters 5, 19 and20.

[0049] Monospecific antiserum was generated according to theabove-mentioned method by immunizing rabbits with the purifiedpolypeptide. The immunogen was mixed with Freund's adjuvant and injectedsubcutaneously into rabbits every second week. Antiserum was obtainedafter a total immunization period of eight weeks, and immunoglobulin wasprepared therefrom as described by N. H. Axelsen, vide supra.

[0050] Ouchterlony double immunodiffusion tests showed no cross reactionbetween the polypeptide encoded by the nucleic acid sequence of theinvention and the well-known alkaline serine proteases Alcalase®,Savinase®, Esperase®, Kazusase® and subtilisin BPN′.

[0051] The nucleic acid sequences of the present invention may beobtained from microorganisms of any genus. For purposes of the presentinvention, the term “obtained from” as used herein in connection with agiven source shall mean that the polypeptide encoded by the nucleic acidsequence is produced by the source or by a cell in which the nucleicacid sequence from the source has been inserted.

[0052] The nucleic acid sequences may be obtained from a bacterialsource. For example, these polypeptides may be obtained from a grampositive bacterium such as a Bacillus strain, e.g., Bacillusalkalophilus, Bacillus amyloliquefaciens, Bacillus brevis, Bacilluscirculans, Bacillus coagulans, Bacillus lautus, Bacillus lentus,Bacillus licheniformis, Bacillus megaterium, Bacillusstearothermophilus, Bacillus subtilis, or Bacillus thuringiensis; or aStreptomyces strain, e.g., Streptomyces lividans or Streptomycesmurinus; or from a gram negative bacterium, e.g., E. coli or Pseudomonassp.

[0053] In a preferred embodiment, the nucleic acid sequences areobtained from Bacillus sp. TY145, and most preferably from Bacillus sp.TY145, NCIMB No. 40339, e.g., the nucleic acid sequence set forth in SEQID NO: 1.

[0054] The preferred microorganism, i.e. Bacillus sp. TY145, isdescribed in WO 92/17577 and was deposited on Dec. 3, 1990 under theaccession number NCIMB No. 40339 as mentioned right above.

[0055] It will be understood that for the aforementioned species, theinvention encompasses both the perfect and imperfect states, and othertaxonomic equivalents, e.g., anamorphs, regardless of the species nameby which they are known. Those skilled in the art will readily recognizethe identity of appropriate equivalents.

[0056] Strains of these species are readily accessible to the public ina number of culture collections, such as the National Collections ofIndustrial & Marine Bacteria Ltd. (NCIMB), the American Type CultureCollection (ATCC), Deutsche Sammiung von Mikroorganismen undZelikulturen GmbH (DSM), Centraalbureau Voor Schimmelcultures (CBS), andAgricultural Research Service Patent Culture Collection, NorthernRegional Research Center (NRRL).

[0057] Furthermore, such nucleic acid sequences may be identified andobtained from other sources including microorganisms isolated fromnature (e.g., soil, composts, water, etc.) using the above-mentionedprobes. Techniques for isolating microorganisms from natural habitatsare well known in the art. The nucleic acid sequence may then be derivedby similarly screening a genomic or cDNA library of anothermicroorganism. Once a nucleic acid sequence encoding a polypeptide hasbeen detected with the probe(s), the sequence may be isolated or clonedby utilizing techniques which are known to those of ordinary skill inthe art (see, e.g., Sambrook et al., 1989, supra).

[0058] The techniques used to isolate or clone a nucleic acid sequenceencoding a polypeptide are known in the art and include isolation fromgenomic DNA, preparation from cDNA, or a combination thereof. Thecloning of the nucleic acid sequences of the present invention from suchgenomic DNA can be effected, e.g., by using the well known polymerasechain reaction (PCR) or antibody screening of expression libraries todetect cloned DNA fragments with shared structural features. See, e.g.,Innis et al., 1990, PCR: A Guide to Methods and Application, AcademicPress, New York. Other nucleic acid amplification procedures such asligase chain reaction (LCR), ligated activated transcription (LAT) andnucleic acid sequence-based amplification (NASBA) may be used. Thenucleic acid sequence may be cloned from a strain of Bacillus, oranother or related organism and thus, for example, may be an allelic orspecies variant of the polypeptide encoding region of the nucleic acidsequence.

[0059] The polypeptides encoded by the isolated nucleic acid sequencesof the present invention have at least 20%, preferably at least 40%,more preferably at least 60%, even more preferably at least 80%, evenmore preferably at least 90%, and most preferably at least 100% of theproteolytic activity of the mature polypeptide of SEQ ID NO: 2.

[0060] Modification of a nucleic acid sequence of the present inventionmay be necessary for the synthesis of polypeptides substantially similarto the polypeptide. The term “substantially similar” to the polypeptiderefers to non-naturally occurring forms of the polypeptide. Thesepolypeptides may differ in some engineered way from the polypeptideisolated from its native source, e.g., variants that differ in specificactivity, thermostability, pH optimum, or the like. The variant sequencemay be constructed on the basis of the nucleic acid sequence presentedas the polypeptide encoding part of SEQ ID NO: 1, e.g., a subsequencethereof, and/or by introduction of nucleotide substitutions which do notgive rise to another amino acid sequence of 5 the polypeptide encoded bythe nucleic acid sequence, but which corresponds to the codon usage ofthe host organism intended for production of the enzyme, or byintroduction of nucleotide substitutions which may give rise to adifferent amino acid sequence. For a general description of nucleotidesubstitution, see, e.g., Ford et al., 1991, Protein Expression andPurification 2: 95-107.

[0061] It will be apparent to those skilled in the art that suchsubstitutions can be made outside the regions critical to the functionof the molecule and still result in an active polypeptide. Amino acidresidues essential to the activity of the polypeptide encoded by theisolated nucleic acid sequence of the invention, and thereforepreferably not subject to substitution, may be identified according toprocedures known in the art, such as site-directed mutagenesis oralanine-scanning mutagenesis (see, e.g., Cunningham and Wells, 1989,Science 244: 1081-1085). In the latter technique, mutations areintroduced at every positively charged residue in the molecule, and theresultant mutant molecules are tested for proteolytic activity toidentify amino acid residues that are critical to the activity of themolecule. Sites of substrate-enzyme interaction can also be determinedby analysis of the three-dimensional structure as determined by suchtechniques as nuclear magnetic resonance analysis, crystallography orphotoaffinity labeling (see, e.g., de Vos et al., 1992, Science 255:306-312; Smith et al., 1992, Journal of Molecular Biology 224: 899-904;Wlodaver et al., 1992, FEBS Letters 309: 59-64).

[0062] A nucleic acid sequence of the present invention may also encodefused polypeptides or cleavable fusion polypeptides in which anotherpolypeptide is fused at the N-terminus or the C-terminus of thepolypeptide or fragment thereof. A fused polypeptide is produced byfusing a nucleic acid sequence (or a portion thereof) encoding anotherpolypeptide to a nucleic acid sequence (or a portion thereof) of thepresent invention. Techniques for producing fusion polypeptides areknown in the art, and include ligating the coding sequences encoding thepolypeptides so that they are in frame and that expression of the fusedpolypeptide is under control of the same promoter(s) and terminator.

[0063] Variants

[0064] In a further aspect, the present invention also relates to avariant of the polypeptide having the amino acid sequence shown as aminoacids 1 to 311 of SEQ ID NO: 1, which comprises at least onemodification compared to amino acids 1 to 311 of SEQ ID NO: 1 and whichhas at least 75% identity with amino acids 1 to 311 of SEQ ID NO: 1.Preferably, the number of modifications is at the most 20, e.g. at themost 15. In an interesting embodiment of the invention, the number ofmodifications is at the most 14, e.g. at the most 13, at the most 12, atthe most 11, at the most 10, at the most 9, at the most 8, at the most7, at the most 6, or at the most 5. In a particular interestingembodiment of the invention, the number of modifications is at the most4, such as at the most 3, e.g. at the most 2, e.g. only onemodification.

[0065] Independent of the exact number of modifications, the number ofmodifications should be so that the variant in question still has atleast 75% identity with amino acids 1 to 311 of SEQ ID NO: 1.Preferably, the variant in question has at least 80% identity, such asat least 90% identity, e.g. at least 95% identity, at least 97% identityor at least 99% identity with amino acids 1 to 311 of SEQ ID NO: 1.

[0066] When used herein, the term “variant” means a polypeptide havingproteolytic activity, which has been produced by an organism, which isexpressing a mutant gene as compared to SEQ ID NO: 1. The mutant gene,from which said variant is produced when expressed in a suitable host,may have been obtained by mutation of the nucleic acid sequencedisclosed in SEQ ID NO: 1. Also, the mutant gene may have been preparedby the DNA shuffling technique.

[0067] In the context of the present invention the term“modification(s)” is intended to mean any chemical modification of thepolypeptide having the amino acid sequence shown as amino acids 1 to 311of SEQ ID NO: 1 as well as genetic manipulation of the DNA encoding thatpolypeptide. The modification(s) can be replacement(s) of the amino acidside chain(s), substitution(s), deletion(s) and/or insertions(s) in orat the amino acid(s) of interest.

[0068] Examples of modifications include, but are not limited to, aminoacid changes which are of a minor nature, that is conservative aminoacid substitutions that do not significantly affect the folding and/oractivity of the protein; small deletions, typically of one to about 30amino acids; small amino- or carboxyl-terminal extensions, such as anamino-terminal methionine residue; a small linker peptide of up to about20-25 residues; or a small extension that facilitates purification bychanging net charge or another function, such as a poly-histidine tract,an antigenic epitope or a binding domain.

[0069] Examples of conservative substitutions are within the group ofbasic amino acids (such as arginine, lysine and histidine), acidic aminoacids (such as glutamic acid and aspartic acid), polar amino acids (suchas glutamine and asparagine), hydrophobic amino acids (such as leucine,isoleucine, methionine and valine), aromatic amino acids (such asphenylalanine, tryptophan and tyrosine), and small amino acids (such asglycine, alanine, serine and threonine). Amino acid substitutions, whichdo not generally alter the specific activity, are known in the art andare described, for example, by H. Neurath and R. L. Hill, 1979, In, TheProteins, Academic Press, New York. The most commonly occurringexchanges are Ala/Ser, Val/Ile, Asp/Glu, Thr/Ser, Ala/Gly, Ala/Thr,Ser/Asn, AlaNal, Ser/Gly, Tyr/Phe, Ala/Pro, Lys/Arg, Asp/Asn, Leu/Ile,LeuNal, Ala/Glu, and Asp/Gly as well as these in reverse.

[0070] The variants of the invention are useful exhibiting excellentwash performance when used in cleaning or detergent composition, inparticular in liquid detergent compositions (vide infra).

[0071] Producing a Variant

[0072] Many methods for cloning a polypeptide and for introducingsubstitutions, deletions and insertions into genes (e.g. subtilasegenes) are well known in the art.

[0073] In general standard procedures for cloning of genes andintroducing insertions (random and/or site directed) into said genes maybe used in order to obtain a variant according to the invention. Forfurther description of suitable techniques reference is made to Sambrooket al. (1989) Molecular cloning: A laboratory manual, Cold Spring HarborLab., Cold Spring Harbor, N.Y.; Ausubel, F. M. et al. (eds.) “Currentprotocols in Molecular Biology”. John Wiley and Sons, 1995; Harwood, C.R., and Cutting, S. M. (eds.) “Molecular Biological Methods forBacillus”. John Wiley and Sons, 1990); and WO 96/34946.

[0074] Further, a variant according to the invention may be constructedby standard techniques for artificial creation of diversity, such as byDNA shuffling of different subtilase genes (WO 95/22625; Stemmer WPC,Nature 370:389-91 (1994)).

[0075] Nucleic Acid Constructs

[0076] The present invention also relates to nucleic acid constructscomprising a nucleic acid sequence of the present invention operablylinked to one or more control sequences, which direct the expression ofthe coding sequence in a suitable host cell under conditions compatiblewith the control sequences. Expression will be understood to include anystep involved in the production of the polypeptide including, but notlimited to, transcription, post-transcriptional modification,translation, post-translational modification, and secretion.

[0077] “Nucleic acid construct” is defined herein as a nucleic acidmolecule, either single- or double-stranded, which is isolated from anaturally occurring gene or which has been modified to contain segmentsof nucleic acid which are combined and juxtaposed in a manner whichwould not otherwise exist in nature. The term nucleic acid construct issynonymous with the term expression cassette when the nucleic acidconstruct contains all the control sequences required for expression ofa coding sequence of the present invention. The term “coding sequence”is defined herein as a portion of a nucleic acid sequence, whichdirectly specifies the amino acid sequence of its protein product. Theboundaries of the coding sequence are generally determined by a ribosomebinding site (prokaryotes) or by the ATG start codon (eukaryotes)located just upstream of the open reading frame at the 5′ end of themRNA and a transcription terminator sequence located just downstream ofthe open reading frame at the 3′ end of the mRNA. A coding sequence caninclude, but is not limited to, DNA, cDNA, and recombinant nucleic acidsequences.

[0078] An isolated nucleic acid sequence encoding a polypeptide of thepresent invention may be manipulated in a variety of ways to provide forexpression of the polypeptide. Manipulation of the nucleic acid sequenceprior to its insertion into a vector may be desirable or necessarydepending on the expression vector. The techniques for modifying nucleicacid sequences utilizing recombinant DNA methods are well known in theart.

[0079] The term “control sequences” is defined herein to include allcomponents that are necessary or advantageous for the expression of apolypeptide of the present invention. Each control sequence may benative or foreign to the nucleic acid sequence encoding the polypeptide.Such control sequences include, but are not limited to, a leader,polyadenylation sequence, propeptide sequence, promoter, signal peptidesequence, and transcription terminator. At a minimum, the controlsequences include a promoter, and transcriptional and translational stopsignals. The control sequences may be provided with linkers for thepurpose of introducing specific restriction sites facilitating ligationof the control sequences with the coding region of the nucleic acidsequence encoding a polypeptide. The term “operably linked” is definedherein as a configuration in which a control sequence is appropriatelyplaced at a position relative to the coding sequence of the DNA sequencesuch that the control sequence directs the expression of a polypeptide.

[0080] The control sequence may be an appropriate promoter sequence, anucleic acid sequence that is recognized by a host cell for expressionof the nucleic acid sequence. The promoter sequence containstranscriptional control sequences, which mediate the expression of thepolypeptide. The promoter may be any nucleic acid sequence which showstranscriptional activity in the host cell of choice including mutant,truncated, and hybrid promoters, and may be obtained from genes encodingextracellular or intracellular polypeptides either homologous orheterologous to the host cell.

[0081] Examples of suitable promoters for directing the transcription ofthe nucleic acid constructs of the present invention, especially in abacterial host cell, are the promoters obtained from the E. coli lacoperon, Streptomyces coelicolor agarase gene (dagA), Bacillus subtilislevansucrase gene (sacB), Bacillus licheniformis alpha-amylase gene(amyL), Bacillus stearothermophilus maltogenic amylase gene (amyM),Bacillus amyloliquefaciens alpha-amylase gene (amyQ), Bacilluslicheniformis penicillinase gene (penP), Bacillus subtilis xyIA and xyIBgenes, and prokaryotic beta-lactamase gene (Villa-Kamaroff et al., 1978,Proceedings of the National Academy of Sciences USA 75: 3727-3731), aswell as the tac promoter (DeBoer et al., 1983, Proceedings of theNational Academy of Sciences USA 80: 21-25). Further 10 promoters aredescribed in “Useful proteins from recombinant bacteria” in ScientificAmerican, 1980, 242: 74-94; and in Sambrook et al., 1989, supra.

[0082] The control sequence may also be a suitable transcriptionterminator sequence, a sequence recognized by a host cell to terminatetranscription. The terminator sequence is operably linked to the 3′terminus of the nucleic acid sequence encoding the polypeptide. Anyterminator that is functional in the host cell of choice may be used inthe present invention.

[0083] The control sequence may also be a suitable leader sequence, anon-translated region of an mRNA that is important for translation bythe host cell. The leader sequence is operably linked to the 5′ terminusof the nucleic acid sequence encoding the polypeptide. Any leadersequence that is functional in the host cell of choice may be used inthe present invention.

[0084] The control sequence may also be a polyadenylation sequence, asequence which is operably linked to the 3′ terminus of the nucleic acidsequence and which, when transcribed, is recognized by the host cell asa signal to add polyadenosine residues to transcribed mRNA.

[0085] Any polyadenylation sequence, which is functional in the hostcell of choice, may be used in the present invention.

[0086] The control sequence may also be a signal peptide coding regionthat codes for an amino acid sequence linked to the amino terminus of apolypeptide and directs the encoded polypeptide into the cell'ssecretory pathway. The 5′ end of the coding sequence of the nucleic acidsequence may inherently contain a signal peptide coding region naturallylinked in translation reading frame with the segment of the codingregion that encodes the secreted polypeptide. Alternatively, the 5′ endof the coding sequence may contain a signal peptide coding region thatis foreign to the coding sequence. The foreign signal peptide codingregion may be required where the coding sequence does not naturallycontain a signal peptide coding region. Alternatively, the foreignsignal peptide coding region may simply replace the natural signalpeptide coding region in order to enhance secretion of the polypeptide.However, any signal peptide coding region that directs the expressedpolypeptide into the secretory pathway of a host cell of choice may beused in the present invention.

[0087] Effective signal peptide coding regions for bacterial host cellsare the signal peptide coding regions obtained from the genes forBacillus NCIB 11837 maltogenic amylase, Bacillus stearothermophilusalpha-amylase, Bacillus licheniformis subtilisin, Bacillus licheniformisbeta-lactamase, Bacillus stearothermophilus neutral proteases (nprT,nprS, nprM), and Bacillus subtilis prsA. Further signal peptides aredescribed by Simonen and Palva, 1993, Microbiological Reviews57:109-137.

[0088] The control sequence may also be a propeptide coding region thatcodes for an amino acid sequence positioned at the amino terminus of apolypeptide. The resultant polypeptide is known as a proenzyme orpropolypeptide (or a zymogen in some cases). A propolypeptide isgenerally inactive and can be converted to a mature active polypeptideby catalytic or autocatalytic cleavage of the propeptide from thepropolypeptide. The propeptide coding region may be obtained from thegenes for Bacillus subtilis alkaline protease (aprE), Bacillus subtilisneutral protease (nprT), Saccharomyces cerevisiae alpha-factor,Rhizomucor miehei aspartic proteinase, and Myceliophthora thermophilalaccase (WO 95/33836).

[0089] Where both signal peptide and propeptide regions are present atthe amino terminus of a polypeptide, the propeptide region is positionednext to the amino terminus of the polypeptide and the signal peptideregion is positioned next to the amino terminus of the propeptideregion.

[0090] It may also be desirable to add regulatory sequences that allowthe regulation of the expression of the polypeptide relative to thegrowth of the host cell. Examples of regulatory systems are those whichcause the expression of the gene to be turned on or off in response to achemical or physical stimulus, including the presence of a regulatorycompound. Regulatory systems in prokaryotic systems include the lac,tac, and trp operator systems. Other examples of regulatory sequencesare those that allow for gene amplification.

[0091] Expression Vectors

[0092] The present invention also relates to recombinant expressionvectors comprising a nucleic acid sequence of the present invention, apromoter, and transcriptional and translational stop signals. Thevarious nucleic acid and control sequences described above may be joinedtogether to produce a recombinant expression vector which may includeone or more convenient restriction sites to allow for insertion orsubstitution of the nucleic acid sequence encoding the polypeptide atsuch sites. Alternatively, the nucleic acid sequence of the presentinvention may be expressed by inserting the nucleic acid sequence or anucleic acid construct comprising the sequence into an appropriatevector for expression. In creating the expression vector, the codingsequence is located in the vector so that the coding sequence isoperably linked with the appropriate control sequences for expression.

[0093] The recombinant expression vector may be any vector (e.g., aplasmid or virus) that can be conveniently subjected to recombinant DNAprocedures and can bring about the expression of the nucleic acidsequence. The choice of the vector will typically depend on thecompatibility of the vector with the host cell into which the vector isto be introduced. The vectors may be linear or closed circular plasmids.

[0094] The vector may be an autonomously replicating vector, i.e., avector that exists as an extrachromosomal entity, the replication ofwhich is independent of chromosomal replication, e.g., a plasmid, anextrachromosomal element, a mini-chromosome, or an artificialchromosome. The vector may contain any means for assuringself-replication. Alternatively, the vector may be one which, whenintroduced into the host cell, is integrated into the genome andreplicated together with the chromosome(s) into which it has beenintegrated. Furthermore, a single vector or plasmid or two or morevectors or plasmids which together contain the total DNA to beintroduced into the genome of the host cell, or a transposon may beused.

[0095] The vectors of the present invention preferably contain one ormore selectable markers that permit easy selection of transformed cells.A selectable marker is a gene the product of which provides for biocideor viral resistance, resistance to heavy metals, prototrophy toauxotrophs, and the like. Examples of bacterial selectable markers arethe dal genes from Bacillus subtilis or Bacillus licheniformis, ormarkers that confer antibiotic resistance such as ampicillin, kanamycin,chloramphenicol or tetracycline resistance.

[0096] The vectors of the present invention preferably contain anelement(s) that permits stable integration of the vector into the hostcell genome or autonomous replication of the vector in the cellindependent of the genome of the cell.

[0097] For integration into the host cell genome, the vector may rely onthe nucleic acid sequence encoding the polypeptide or any other elementof the vector for stable integration of the vector into the genome byhomologous or non-homologous recombination. Alternatively, the vectormay contain additional nucleic acid sequences for directing integrationby homologous recombination into the genome of the host cell. Theadditional nucleic acid sequences enable the vector to be integratedinto the host cell genome at a precise location(s) in the chromosome(s).To increase the likelihood of integration at a precise location, theintegrational elements should preferably contain a sufficient number ofnucleic acids, such as 100 to 1,500 base pairs, preferably 400 to 1,500base pairs, and most preferably 800 to 1,500 base pairs, which arehighly homologous with the corresponding target sequence to enhance theprobability of homologous recombination. The integrational elements maybe any sequence that is homologous with the target sequence in thegenome of the host cell. Furthermore, the integrational elements may benon-encoding or encoding nucleic acid sequences. On the other hand, thevector may be integrated into the genome of the host cell bynon-homologous recombination.

[0098] For autonomous replication, the vector may further comprise anorigin of replication enabling the vector to replicate autonomously inthe host cell in question. Examples of bacterial origins of replicationare the origins of replication of plasmids pBR322, pUC19, pACYC177, andpACYC184 permitting replication in E. coli, and pUB110, pE194, pTA1060,and pAMβ1 permitting replication in Bacillus. The origin of replicationmay be one having a mutation which makes its functioningtemperature-sensitive in the host cell (see, e.g., Ehrlich, 1978,Proceedings of the National Academy of Sciences USA 75: 1433).

[0099] More than one copy of a nucleic acid sequence of the presentinvention may be inserted into the host cell to increase production ofthe gene product. An increase in the copy number of the nucleic acidsequence can be obtained by integrating at least one additional copy ofthe sequence into the host cell genome or by including an amplifiableselectable marker gene with the nucleic acid sequence where cellscontaining amplified copies of the selectable marker gene, and therebyadditional copies of the nucleic acid sequence, can be selected for bycultivating the cells in the presence of the appropriate selectableagent.

[0100] The procedures used to ligate the elements described above toconstruct the recombinant expression vectors of the present inventionare well known to one skilled in the art (see, e.g., Sambrook et al.,1989, supra).

[0101] Host Cells

[0102] The present invention also relates to recombinant host cells,comprising a nucleic acid sequence of the invention, which areadvantageously used in the recombinant production of the polypeptides. Avector comprising a nucleic acid sequence of the present invention isintroduced into a host cell so that the vector is maintained as achromosomal integrant or as a self-replicating extra-chromosomal vectoras described earlier. The term “host cell” encompasses any progeny of aparent cell that is not identical to the parent cell due to mutationsthat occur during replication. The choice of a host cell will to a largeextent depend upon the gene encoding the polypeptide and its source.

[0103] The host cell may be a unicellular microorganism, e.g., aprokaryote, or a non-unicellular microorganism, e.g., a eukaryote.

[0104] Useful unicellular cells are bacterial cells such as grampositive bacteria including, but not limited to, a Bacillus cell, e.g.,Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillus brevis,Bacillus circulans, Bacillus clausii, Bacillus coagulans, Bacilluslautus, Bacillus lentus, Bacillus licheniformis, Bacillus megaterium,Bacillus stearothermophilus, Bacillus subtilis, and Bacillusthuringiensis; or a Streptomyces cell, e.g., Streptomyces lividans orStreptomyces murinus, or gram negative bacteria such as E. coli andPseudomonas sp. In a preferred embodiment, the bacterial host cell is aBacillus lentus, Bacillus licheniformis, Bacillus stearothermophilus orBacillus subtilis cell. In another preferred embodiment, the Bacilluscell is an alkalophilic Bacillus.

[0105] The introduction of a vector into a bacterial host cell may, forinstance, be effected by protoplast transformation (see, e.g., Chang andCohen, 1979, Molecular General Genetics 168: 111-115), using competentcells (see, e.g., Young and Spizizen, 1961, Journal of Bacteriology 81:823-829, or Dubnau and Davidoff-Abelson, 1971, Journal of MolecularBiology 56: 209-221), electroporation (see, e.g., Shigekawa and Dower,1988, Biotechniques 6: 742-751), or conjugation (see, e.g., Koehler andThorne, 1987, Journal of Bacteriology 169: 5771-5278).

[0106] Methods of Production

[0107] The present invention also relates to methods for producing apolypeptide comprising (a) cultivating a host cell of the inventionunder conditions conducive for production of the polypeptide; and (b)recovering the polypeptide.

[0108] In the production methods of the present invention, the cells arecultivated in a nutrient medium suitable for production of thepolypeptide using methods known in the art. For example, the cell may becultivated by shake flask cultivation, small-scale or large-scalefermentation (including continuous, batch, fed-batch, or solid statefermentations) in laboratory or industrial fomenters performed in asuitable medium and under conditions allowing the polypeptide to beexpressed and/or isolated. The cultivation takes place in a suitablenutrient medium comprising carbon and nitrogen sources and inorganicsalts, using procedures known in the art. Suitable media are availablefrom commercial suppliers or may be prepared according to publishedcompositions (e.g., in catalogues of the American Type CultureCollection). If the polypeptide is secreted into the nutrient medium,the polypeptide can be recovered directly from the medium. If thepolypeptide is not secreted, it can be recovered from cell lysates.

[0109] The polypeptides may be detected using methods known in the artthat are specific for the polypeptides. These detection methods mayinclude use of specific antibodies, formation of an enzyme product, ordisappearance of an enzyme substrate. For example, an enzyme assay maybe used to determine the activity of the polypeptide as describedherein.

[0110] The resulting polypeptide may be recovered by methods known inthe art. For example, the polypeptide may be recovered from the nutrientmedium by conventional procedures including, but not limited to,centrifugation, filtration, extraction, spray-drying, evaporation, orprecipitation.

[0111] The polypeptides may be purified by a variety of procedures knownin the art including, but not limited to, chromatography (e.g., ionexchange, affinity, hydrophobic, chromatofocusing, and size exclusion),electrophoretic procedures (e.g., preparative isoelectric focusing),differential solubility (e.g., ammonium sulfate precipitation),SDS-PAGE, or extraction (see, e.g., Protein Purification, J. -C. Jansonand Lars Ryden, editors, VCH Publishers, New York, 1989).

[0112] Uses

[0113] The polypeptides (including the variants) having proteolyticactivity encoded by the nucleic acid sequences of the present inventionare useful as ingredients in detergent compositions. In addition to thepolypeptides having proteolytic activity, described herein, suchdetergent compositions typically comprise one or more surfactants, whichmay be of an anionic, non-ionic, cat-ionic, amphoteric or zwitter-ionictype, or a mixture of these. Typical examples of anionic surfactants arelinear alkyl benzene sulfonates (LAS), alkyl sulfates (AS), alpha olefinsulfonates (AOS), alcohol ethoxy sulfates (AES) and alkali metal saltsof natural fatty acids. Examples of non-ionic surfactants are alkylpolyethylene glycol ethers, nonylphenol polyethylene glycol ethers,fatty acids esters of sucrose and glucose, and esters of polyethoxylatedalkyl glucoside.

[0114] The detergent composition may also contain other detergentingredients known in the art such as builders, bleaching agents, bleachactivators, anti-corrosion agents, sequestering agents, antisoil-redeposition agents, perfumes, stabilizers for the enzymes andbleaching agents, formulations aids, optical brighteners, foam boosters,chelating agents, fillers, fabric softeners, etc. The detergentcomposition may be formulated substantially as described in J. Falbe:Surfactants in Consumer Products. Theory, Technology and Application;Springer Verlag 1987, in particular the section entitled “Frameformulations for liquid/powder heavy-duty detergents”.

[0115] It is at present contemplated that the detergent composition maycontain the enzyme preparation in an amount corresponding to 0.0005-0.5CPU of the proteolytic enzyme per liter of washing liquor.

[0116] The detergent compositions can be formulated in any convenientform, such as powders, liquids, etc.

[0117] The detergent composition of the invention may advantageouslyinclude one or more other enzymes, e.g. lipases, amylases, cellulases,oxidases, peroxidases and/or other proteases, conventionally included indetergent compositions.

[0118] The polypeptide having proteolytic activity may be included in adetergent composition by adding separate additives containing thedetergent protease, or by adding a combined additive comprisingdifferent detergent enzymes.

[0119] The additive of the invention can be formulated e.g. asgranulates, liquids, slurries, etc. Preferred detergent additiveformulations are non-dusting granulates, liquids, in particularstabilized liquids, slurries, or protected enzymes. Dust free granulatesmay be produced e.g. according to GB Patent Publication No. 1,362,365 orU.S. Pat. No. 4,106,991, and may optionally be coated by methods knownin the art. The detergent enzymes may be mixed before or aftergranulation. Liquid enzyme preparations may, for instance, be stabilizedby adding a polyol such as e.g. propylene glycol, a sugar or sugaralcohol, lactic acid or boric acid, according to established methods.Other enzyme stabilizers are well known in the art. Protected enzymesmay be prepared according to the method disclosed in EP PatentPublication No. 238,216.

[0120] The present invention is further described by the followingexamples, which should not be construed as limiting the scope of theinvention.

EXAMPLES

[0121] General Molecular Biology Methods:

[0122] Unless otherwise mentioned the DNA manipulations andtransformations were performed using standard methods of molecularbiology (Sambrook et al. (1989) Molecular cloning: A laboratory manual,Cold Spring Harbor lab., Cold Spring Harbor, N.Y.; Ausubel, F. M. et al.(eds.) “Current protocols in Molecular Biology”. John Wiley and Sons,1995; Harwood, C. R., and Cutting, S. M. (eds.) “Molecular BiologicalMethods for Bacillus”. John Wiley and Sons, 1990). Enzymes for DNAmanipulations were used according to the specifications of thesuppliers.

[0123] Unless otherwise mentioned all enzymes for DNA manipulations,such as e.g. restriction endonucleases, ligases etc., are obtained fromNew England Biolabs, Inc.

[0124] Chemicals used as buffers and substrates were commercial productsof at least reagent grade.

Example 1 Cultivation of Bacillus sp. TY145

[0125] Bacillus sp. TY145 was cultivated at 25° C. on a rotary shakingtable (300 r.p.m.) in 500 ml baffled Erlenmeyer flasks containing 100 mlof medium of the following composition (per liter): Potato starch 100 g Ground barley 50 g Soybean flour 20 g Na₂HPO₄ × 2H₂O  9 g Pluronic ® 0.1g  Sodium caseinate 10 g

[0126] The starch in the medium is liquefied with a-amylase and themedium is sterilized by heating at 120° C. for 45 minutes.

[0127] After sterilization the pH of the medium is adjusted to 9.0 byaddition of 10 ml of a 1 M solution of sodium bicarbonate.

[0128] After 5 days of incubation the proteolytic activity of theculture was determined using the method described herein. Aftercultivation, the enzyme activity of the broth was 10 CPU/I.

[0129] After separation of the solid material the protease was purifiedby a conventional chromatographic method.

[0130] Yield from 1 liter of culture broth was 50 ml with 57 CPU/I.Purity was more than 90% as judged by SDS-PAGE.

Example 2 Wash Performance

[0131] The wash performance tests were performed on grass soiling oncotton, in a model wash system at 20° C., isothermically for 10 minutes.

[0132] 2.0 g/l of a commercial American type liquid detergent was usedin the tests. The detergent did not contain any enzymes prior to theaddition of the protease of the invention. The detergent was dissolvedin approx. 6°dH (German Hardness) water, and the pH was measured toapprox. 8. The textile/wash liquor ratio was approximately 6 g textileper liter of detergent solution. The enzyme preparation according toExample 1 was used at enzyme protein concentrations of 0.01; 0.04; 0.08;0.16, and 0.5 CPU/liter.

[0133] Subsequent to washing, the fabric was rinsed in running tap waterfor 25 minutes and air-dried. The wash performance was determined by thechange (ΔR) of the remission (%R) at 460 nm measured on a DatacolorElrephometer 2000, ΔR being the remission after wash with thepolypeptide having the amino acid sequence shown as amino acids 1 to 311of SEQ ID NO: 2 minus the remission after wash with no protease added.

[0134] The test results are shown in the table below. EnzymeConcentration ΔR 0.01 CPU/l 4.0 0.04 CPU/l 6.7 0.08 CPU/l 8.7 0.16 CPU/l9.7 0.50 CPU/l 11.2 

[0135] The differential remission values (AR) show that the polypeptidehaving the amino acid sequence shown as amino acids 1 to 311 of SEQ IDNO: 2 possesses a good washability.

Example 3 Stability in Detergents

[0136] The stability of the polypeptide having the amino acid sequenceshown as amino acids 1 to 311 of SEQ ID NO: 2 was tested in the presenceof detergents. The detergents used in this test were an American typepowder detergent and an American type liquid detergent.

[0137] The residual activity was determined after 60 minutes at 40° C.Enzyme dosage was 0.3 CPU/I. Residual activity Powder detergent: 0.9 g/l100% Liquid detergent: 2.0 g/l  95%

[0138] This experiment shows that the polypeptide having the amino acidsequence shown as amino acids 1 to 311 of SEQ ID NO: 2 is stable indetergents under wash conditions.

Example 4 Wash Performance in US Liquid Detergent Compared to Savinase®

[0139] Wash conditions: Detergent dosage: 8 g/l Wash temperature: 30° C.Wash time: 12 minutes Water hardness: 6° dH (Ca²⁺:Mg²⁺ = 2:1) pH: Notadjusted (8.2 before wash) Enzyme concentrations: 1.25, 2.5, 5, 10, 30nM Test system: 150 ml glass beakers with a stirring rod Textile/volume:5 textile pieces (Ø 2.5 cm) in 50 ml detergent Test material: EMPA117(blood, ink, milk) and CS8 (milk)

[0140] Detergents:

[0141] The detergents used were obtained from supermarkets in the USA(Tide Mountain Spring, Deep Clean Formula 1999 P&G 40084959). Prior touse all enzymatic activity in the detergent was inactivated by microwavetreatment.

[0142] Swatches:

[0143] The swatches used were EMPA117, obtained from EMPATestmaterialen, Movenstrasse 12, CH-9015. St. Gall, Switzerland, andCS8, obtained from CFT Center For Testmaterials, Hoekerstraat 12, 3133KR Vlaardingen, The Netherlands.

[0144] Reflectance:

[0145] Measurement of reflectance (R) on the test materials was done at460 nm using a Macbeth ColorEye 7000 photometer. The measurements weredone in accordance with the manufacturer's protocol.

[0146] Evaluation:

[0147] The evaluation of the wash performance of a protease isdetermined by the improvement factor of the protease investigated.

[0148] The improvement factor, IF_(dose/response), is defined as theratio between the slopes of the wash performance curves for a detergentcontaining the proteases to be investigated and the same detergentcontaining a reference protease (in this case Savinase®) at theasymptotic concentration of the protease goes to zero, i.e.IF_(dose/response)=a/a_(ref).

[0149] The wash performance is calculated according to the below formulaI:

R=R ₀+(a·ΔR _(max) ·c)/(ΔR _(max) +a·c)  (I)

[0150] where

[0151] R is the wash performance in reflectance units; R₀ is theintercept of the fitted curve with the y-axis (blind); a is the slope ofthe fitted curve as c→0; c is the enzyme concentration; and ΔR_(max) isthe theoretical maximal wash effect as c→∞. Results: Protease SwatchIF_(dose/response) Savinase ® EMPA117 1   SEQ ID NO: 2 EMPA117 4.7Savinase ® CS8 1   SEQ ID NO: 2 CS8 3.1

[0152] As it appears, the protease having the amino acid sequence shownas amino acids 1-311 of SEQ ID NO: 2 exhibits improved wash performanceas compared to Savinase®.

Example 5 Determination of Sequence

[0153] The clone of interest was selected as protease positive fromscreening of a Bacillus sp. TY145 NCIMB no. 40339 gene library on agarplate with skim milk. The clone contains an approximately 4.2 Kb insert.The insert was PCR amplified using Expand Long Template PCR System(Roche) according to manufactures instruction. Transposon insertion wasdone directly on the PCR product using the GPS-1, Genome PrimingSystems, from New England Biolabs Inc. The transposon inserted DNA poolwas digested with Hind III (New England Biolabs inc.) and ligated intoHind III digested pZErO 2.0 (Invitrogen). The DNA was transformed intoE. coli by standard procedures. Qiagen (Qiagen, USA) purified plasmidDNA from chloramphenicol and kanamycin resistant clones were isolatedfrom E. coli. Inserts of the isolated clones were sequenced with M13forward and reverse primers, PrimerS (New England Biolabs inc.) andPrimerN (New England Biolabs inc.), using the Taq deoxy terminal cyclesequencing kit (Perkin Elmer, USA) and an Applied Biosystems ABI PRISM™377 DNA Sequencer according to the manufacturers instructions. Bycombining the DNA sequences obtained by the above mention method, theDNA sequence of the original insert in PRT1313 was determined. DNAsequence analysis revealed an open reading frame, ORF, containing theDNA sequence for the Alkaline Bacillus protease TY145 (SEQ ID NO: 1).

Example 6 Expression of the apr Protease from Bacillus sp. TY145 inBacillus subtilis

[0154] The AprTY145 protease gene was cloned into a derivative ofpSX120* (WO91/09129) and transformed to B. subtilis DN497 as an in framefusion to the aprH309 signal sequence and flanked C-terminal by theterminator of aprH309.

[0155] The apr protease gene was isolated by PCR from chromosomal DNA ofBacillus sp. TY145 (NCIMP No. 40339; WO 92/17577) by the specificprimers pep81 (SEQ ID NO: 3) and pep86 (SEQ ID NO: 4).

[0156] The pro-region and the coding region for the mature apr proteasewere fused in frame with the signal sequence of aprH309 (WO 89/06279)via a unique Cla1 site. The aprTY145 PCR sequence was inserted into thepSX222 vector as a Cla1 Mlu1 fragment replacing the original pro andmature sequence of the aprH309 protease. (the sequence of fusedprotease+the terminator region of aprH309 from the ATG start to BamHIbehind the aprH309 terminator is set forth in SEQ ID NO: 5)

[0157] After ligation the DNA was transformed to competent B. subtiliscells (DN497, delta aprE, delta nprE) and transformants resistanttowards 10 microgram/ml chloramphenicol was isolated. (Transformation ofB. subtilis was performed as described by Dubnau et al., 1971, J. Mol.Biol. 56, pp. 209-221.)

[0158] The DNA sequence obtained from the transformants confirms theinsert to be the correct fusion of the aprH309 and the aprTY145protease.

[0159] The Mw of the AprTY145 protease recovered from this transformantwas analysed by mass spectoscopy and the Mw of 31784 Dalton is exactlyidentical to the Mw of the Apr protease obtained from the B. sp. TY145.

[0160] The pSX222 vector used in this experiment is a derivative ofpSX120 -In pSX222 the original aprH309 gene is replaced by a partiallysynthetic DNA sequence of the AprH309 protease. The synthetic gene hasadditional restriction sites integrated in the coding sequence and ashorter terminator sequence compared to the aprH309 gene in pSX120.

1 5 1 1306 DNA Bacillus sp. CDS (50)..(1303) 1 aaatataata ttagcgaaagagaaattaca atttgagagg agaaatggg atg aag 55 Met Lys aaa aga aga gca tttgca gcc aca tta ctc agt att acg atg gga tta 103 Lys Arg Arg Ala Phe AlaAla Thr Leu Leu Ser Ile Thr Met Gly Leu -105 -100 -95 -90 tcc gta ttttca aca gga gca ctt gca aaa gac aaa gtt gag gta aag 151 Ser Val Phe SerThr Gly Ala Leu Ala Lys Asp Lys Val Glu Val Lys -85 -80 -75 gaa caa gattca tat cgt gtg cta atc aaa gca cca act aca tca atc 199 Glu Gln Asp SerTyr Arg Val Leu Ile Lys Ala Pro Thr Thr Ser Ile -70 -65 -60 agt act tttcaa tca caa tac gat gtc cgt tgg gat ttt ggc aaa gag 247 Ser Thr Phe GlnSer Gln Tyr Asp Val Arg Trp Asp Phe Gly Lys Glu -55 -50 -45 gga ttt acaaca gat gtt gat gcc aaa cag ctc caa acg ctt caa agc 295 Gly Phe Thr ThrAsp Val Asp Ala Lys Gln Leu Gln Thr Leu Gln Ser -40 -35 -30 aac aaa gacatt caa att cag aag gta aat gaa atg aca gta gaa act 343 Asn Lys Asp IleGln Ile Gln Lys Val Asn Glu Met Thr Val Glu Thr -25 -20 -15 -10 gtt acaaca gaa aag gcg gaa gtg acg gcg gta cca agt aca caa acc 391 Val Thr ThrGlu Lys Ala Glu Val Thr Ala Val Pro Ser Thr Gln Thr -5 -1 1 5 cct tggggc ata aag tca att tat aat gat caa tca att aca aaa aca 439 Pro Trp GlyIle Lys Ser Ile Tyr Asn Asp Gln Ser Ile Thr Lys Thr 10 15 20 act gga ggcagc gga att aag gta gct gtt tta gat aca ggg gtt tat 487 Thr Gly Gly SerGly Ile Lys Val Ala Val Leu Asp Thr Gly Val Tyr 25 30 35 aca agc cat ttagat tta gct ggt tct gcc gag caa tgc aag gat ttt 535 Thr Ser His Leu AspLeu Ala Gly Ser Ala Glu Gln Cys Lys Asp Phe 40 45 50 55 acc caa tct aatcct tta gta gat ggt tca tgc acc gat cgc caa ggg 583 Thr Gln Ser Asn ProLeu Val Asp Gly Ser Cys Thr Asp Arg Gln Gly 60 65 70 cat ggt aca cat gttgcc gga act gta ttg gcg cat gga ggc agt aat 631 His Gly Thr His Val AlaGly Thr Val Leu Ala His Gly Gly Ser Asn 75 80 85 gga caa ggc gtt tac ggggtg gct ccg caa gcg aaa cta tgg gca tat 679 Gly Gln Gly Val Tyr Gly ValAla Pro Gln Ala Lys Leu Trp Ala Tyr 90 95 100 aaa gta tta gga gat aacggc agc gga tac tct gat gat att gca gca 727 Lys Val Leu Gly Asp Asn GlySer Gly Tyr Ser Asp Asp Ile Ala Ala 105 110 115 gct atc aga cat gta gctgat gaa gct tca cgt aca ggt tcc aaa gta 775 Ala Ile Arg His Val Ala AspGlu Ala Ser Arg Thr Gly Ser Lys Val 120 125 130 135 gta att aat atg tcgcta ggt tca tct gcc aag gat tca ttg att gct 823 Val Ile Asn Met Ser LeuGly Ser Ser Ala Lys Asp Ser Leu Ile Ala 140 145 150 agt gca gta gat tatgca tat gga aaa ggt gta tta atc gtt gct gcg 871 Ser Ala Val Asp Tyr AlaTyr Gly Lys Gly Val Leu Ile Val Ala Ala 155 160 165 gct ggt aat agt gggtca ggc agc aat aca atc ggc ttt cct ggc ggg 919 Ala Gly Asn Ser Gly SerGly Ser Asn Thr Ile Gly Phe Pro Gly Gly 170 175 180 ctt gta aat gca gtggca gta gcg gca ttg gag aat gtt cag caa aat 967 Leu Val Asn Ala Val AlaVal Ala Ala Leu Glu Asn Val Gln Gln Asn 185 190 195 gga act tat cga gtagct gat ttc tca tct aga ggg aat ccg gca act 1015 Gly Thr Tyr Arg Val AlaAsp Phe Ser Ser Arg Gly Asn Pro Ala Thr 200 205 210 215 gct gga gat tatatc att caa gag cgt gat att gaa gtt tca gct ccg 1063 Ala Gly Asp Tyr IleIle Gln Glu Arg Asp Ile Glu Val Ser Ala Pro 220 225 230 gga gca agt gtagag tct aca tgg tac act ggc ggt tat aat acg atc 1111 Gly Ala Ser Val GluSer Thr Trp Tyr Thr Gly Gly Tyr Asn Thr Ile 235 240 245 agc ggt aca tcaatg gct aca cct cat gta gct ggg tta gct gct aaa 1159 Ser Gly Thr Ser MetAla Thr Pro His Val Ala Gly Leu Ala Ala Lys 250 255 260 atc tgg tca gcgaat act tca tta agt cat agc caa ctg cgc aca gaa 1207 Ile Trp Ser Ala AsnThr Ser Leu Ser His Ser Gln Leu Arg Thr Glu 265 270 275 ttg caa aat cgcgct aaa gta tat gat att aaa ggt ggt atc gga gcc 1255 Leu Gln Asn Arg AlaLys Val Tyr Asp Ile Lys Gly Gly Ile Gly Ala 280 285 290 295 gga aca ggtgac gat tat gca tca ggg ttc gga tat cca aga gta aaa 1303 Gly Thr Gly AspAsp Tyr Ala Ser Gly Phe Gly Tyr Pro Arg Val Lys 300 305 310 taa 1306 2418 PRT Bacillus sp. 2 Met Lys Lys Arg Arg Ala Phe Ala Ala Thr Leu LeuSer Ile Thr Met -105 -100 -95 Gly Leu Ser Val Phe Ser Thr Gly Ala LeuAla Lys Asp Lys Val Glu -90 -85 -80 Val Lys Glu Gln Asp Ser Tyr Arg ValLeu Ile Lys Ala Pro Thr Thr -75 -70 -65 -60 Ser Ile Ser Thr Phe Gln SerGln Tyr Asp Val Arg Trp Asp Phe Gly -55 -50 -45 Lys Glu Gly Phe Thr ThrAsp Val Asp Ala Lys Gln Leu Gln Thr Leu -40 -35 -30 Gln Ser Asn Lys AspIle Gln Ile Gln Lys Val Asn Glu Met Thr Val -25 -20 -15 Glu Thr Val ThrThr Glu Lys Ala Glu Val Thr Ala Val Pro Ser Thr -10 -5 -1 1 5 Gln ThrPro Trp Gly Ile Lys Ser Ile Tyr Asn Asp Gln Ser Ile Thr 10 15 20 Lys ThrThr Gly Gly Ser Gly Ile Lys Val Ala Val Leu Asp Thr Gly 25 30 35 Val TyrThr Ser His Leu Asp Leu Ala Gly Ser Ala Glu Gln Cys Lys 40 45 50 Asp PheThr Gln Ser Asn Pro Leu Val Asp Gly Ser Cys Thr Asp Arg 55 60 65 Gln GlyHis Gly Thr His Val Ala Gly Thr Val Leu Ala His Gly Gly 70 75 80 85 SerAsn Gly Gln Gly Val Tyr Gly Val Ala Pro Gln Ala Lys Leu Trp 90 95 100Ala Tyr Lys Val Leu Gly Asp Asn Gly Ser Gly Tyr Ser Asp Asp Ile 105 110115 Ala Ala Ala Ile Arg His Val Ala Asp Glu Ala Ser Arg Thr Gly Ser 120125 130 Lys Val Val Ile Asn Met Ser Leu Gly Ser Ser Ala Lys Asp Ser Leu135 140 145 Ile Ala Ser Ala Val Asp Tyr Ala Tyr Gly Lys Gly Val Leu IleVal 150 155 160 165 Ala Ala Ala Gly Asn Ser Gly Ser Gly Ser Asn Thr IleGly Phe Pro 170 175 180 Gly Gly Leu Val Asn Ala Val Ala Val Ala Ala LeuGlu Asn Val Gln 185 190 195 Gln Asn Gly Thr Tyr Arg Val Ala Asp Phe SerSer Arg Gly Asn Pro 200 205 210 Ala Thr Ala Gly Asp Tyr Ile Ile Gln GluArg Asp Ile Glu Val Ser 215 220 225 Ala Pro Gly Ala Ser Val Glu Ser ThrTrp Tyr Thr Gly Gly Tyr Asn 230 235 240 245 Thr Ile Ser Gly Thr Ser MetAla Thr Pro His Val Ala Gly Leu Ala 250 255 260 Ala Lys Ile Trp Ser AlaAsn Thr Ser Leu Ser His Ser Gln Leu Arg 265 270 275 Thr Glu Leu Gln AsnArg Ala Lys Val Tyr Asp Ile Lys Gly Gly Ile 280 285 290 Gly Ala Gly ThrGly Asp Asp Tyr Ala Ser Gly Phe Gly Tyr Pro Arg 295 300 305 Val Lys 3103 45 DNA Artificial Sequence Primer 3 gttcatcgat cgcatcggct gcacttgcaaaagacaaagt tgagg 45 4 34 DNA Artificial Sequence Primer 4 atgcaggcgttattttactc ttggatatcc gaac 34 5 1330 DNA Artificial Sequence Synthetic 5atgaagaaac cgttggggaa aattgtcgca agcaccgcac tactcatttc tgttgctttt 60agttcatcga tcgcatcggc tgcacttgca aaagacaaag ttgaggtaaa ggaacaagat 120tcatatcgtg tgctaatcaa agcaccaact acatcaatca gtacttttca atcacaatac 180gatgtccgtt gggattttgg caaagaggga tttacaacag atgttgatgc caaacagctc 240caaacgcttc aaagcaacaa agacattcaa attcagaagg taaatgaaat gacagtagaa 300actgttacaa cagaaaaggc ggaagtgacg gcggtaccaa gtacacaaac cccttggggc 360ataaagtcaa tttataatga tcaatcaatt acaaaaacaa ctggaggcag cggaattaag 420gtagctgttt tagatacagg ggtttataca agccatttag atttagctgg ttctgccgag 480caatgcaagg attttaccca atctaatcct ttagtagatg gttcatgcac cgatcgccaa 540gggcatggta cacatgttgc cggaactgta ttggcgcatg gaggcagtaa tggacaaggc 600gtttacgggg tggctccgca agcgaaacta tgggcatata aagtattagg agataacggc 660agcggatact ctgatgatat tgcagcagct atcagacatg tagctgatga agcttcacgt 720acaggttcca aagtagtaat taatatgtcg ctaggttcat ctgccaagga ttcattgatt 780gctagtgcag tagattatgc atatggaaaa ggtgtattaa tcgttgctgc ggctggtaat 840agtgggtcag gcagcaatac aatcggcttt cctggcgggc ttgtaaatgc agtggcagta 900gcggcattgg agaatgttca gcaaaatgga acttatcgag tagctgattt ctcatctaga 960gggaatccgg caactgctgg agattatatc attcaagagc gtgatattga agtttcagct 1020ccgggagcaa gtgtagagtc tacatggtac actggcggtt ataatacgat cagcggtaca 1080tcaatggcta cacctcatgt agctgggtta gctgctaaaa tctggtcagc gaatacttca 1140ttaagtcata gccaactgcg cacagaattg caaaatcgcg ctaaagtata tgatattaaa 1200ggtggtatcg gagccggaac aggtgacgat tatgcatcag ggttcggata tccaagagta 1260aaataacgcg ttaatcaata aaaaaacgct gtgcggttaa agggcacagc gtttttttgt 1320gtatggatcc 1330

1. An isolated nucleic acid sequence encoding a polypeptide havingproteolytic activity, selected from the group consisting of: (a) anucleic acid sequence encoding a polypeptide having an amino acidsequence which has at least 75% identity with amino acids 1 to 311 ofSEQ ID NO: 2; (b) a nucleic acid sequence having at least 70% identitywith nucleotides 371 to 1303 of SEQ ID NO: 1; (c) a nucleic acidsequence, which hybridizes under low stringency conditions with (i) thenucleic acid sequence of SEQ ID NO: 1, (ii) a subsequence of (i) of atleast 100 nucleotides, or (iii) a complementary strand of (i) or (ii);(d) an allelic variant of (a), (b), or (c); and (e) a subsequence of(a), (b), (c), or (d), wherein the subsequence encodes a polypeptidefragment which has proteolytic activity:
 2. The nucleic acid sequence ofclaim 1, which encodes a polypeptide having an amino acid sequence whichhas at least 75% identity with amino acids 1 to 311 of SEQ ID NO:
 2. 3.The nucleic acid sequence of claim 2, which encodes a polypeptide havingan amino acid sequence which has at least 80% identity with amino acids1 to 311 of SEQ ID NO:
 2. 4. The nucleic acid sequence of claim 3, whichencodes a polypeptide having an amino acid sequence which has at least90% identity with amino acids 1 to 311 of SEQ ID NO:
 2. 5. The nucleicacid sequence of claim 4, which encodes a polypeptide having an aminoacid sequence which has at least 95% identity with amino acids 1 to 311of SEQ ID NO:
 2. 6. The nucleic acid sequence of claim 5, which encodesa polypeptide having an amino acid sequence which has at least 99%identity with amino acids 1 to 311 of SEQ ID NO:
 2. 7. The nucleic acidsequence of claim 1, which encodes a polypeptide comprising the aminoacids 1 to 311 of SEQ ID NO:
 2. 8. The nucleic acid sequence of claim 7,which encodes a polypeptide consisting of the amino acids 1 to 311 ofSEQ ID NO:
 2. 9. The nucleic acid sequence of claim 1 which has at least70% identity with nucleotides 371 to 1303 of SEQ ID NO:
 1. 10. Thenucleic acid sequence of claim 9 which has at least 80% identity withnucleotides 371 to 1303 of SEQ ID NO:
 1. 11. The nucleic acid sequenceof claim 10 which has at least 90% identity with nucleotides 371 to 1303of SEQ ID NO:
 1. 12. The nucleic acid sequence of claim 11 which has atleast 95% identity with nucleotides 371 to 1303 of SEQ ID NO:
 1. 13. Thenucleic acid sequence of claim 12, which has at least 99% identity withnucleotides 371 to 1303 of SEQ ID NO:
 1. 14. The nucleic acid sequenceof claim 1, which comprises the nucleotides 371 to 1303 of SEQ ID NO: 1.15. The nucleic acid sequence of claim 14, which consists of nucleotides371 to 1303 of SEQ ID NO:
 1. 16. The nucleic acid sequence of claim 1,wherein the nucleic acid sequence hybridizes under low stringencyconditions with (i) the nucleic acid sequence of SEQ ID NO:1, (ii) asubsequence of (i) of at least 100 nucleotides, or (iii) a complementarystrand of (i) or (ii).
 17. The nucleic acid sequence of claim 16,wherein the nucleic acid sequence hybridizes under medium stringencyconditions.
 18. The nucleic acid sequence of claim 17, wherein thenucleic acid sequence hybridizes under high stringency conditions. 19.The nucleic acid sequence of claim 1, which encodes a polypeptide whichhas at least 20% of the proteolytic activity of amino acids 1 to 311 ofSEQ ID NO:
 2. 20. A nucleic acid construct comprising the nucleic acidsequence of any of claims 1-19 operably linked to one or more controlsequences, which direct the production of the polypeptide in a suitableexpression host.
 21. A recombinant expression vector comprising thenucleic acid construct of claim 20, a promoter, and transcriptional andtranslational stop signals.
 22. A recombinant host cell comprising thenucleic acid construct of claim
 20. 23. The host cell of claim 22, whichis a bacterium.
 24. The host cell of claim 23, which is a Bacillus. 25.The host cell of claim 24, which is a Bacillus subtilis.
 26. A methodfor producing a polypeptide having proteolytic activity, the methodcomprising (a) cultivating the host cell of claim 22 under conditionsconducive to the production of the polypeptide; and (b) recovering thepolypeptide.
 27. A variant of the polypeptide having the amino acidsequence shown as amino acids 1 to 311 of SEQ ID NO:2, which comprisesat least one modification compared to amino acids 1 to 311 of SEQ IDNO:2 and which has at least 75% identity with amino acids 1 to 311 ofSEQ ID NO:1.
 28. A detergent composition comprising the variant of claim27.
 29. A detergent composition according to claim 28, wherein thedetergent composition is a liquid detergent composition.
 30. A detergentcomposition according to claims 28-29, which additionally comprises acellulase, lipase, cutinase, oxidoreductase, amylase, another protease,or a mixture thereof.